Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Aug 26, 2020; 12(8): 841-856
Published online Aug 26, 2020. doi: 10.4252/wjsc.v12.i8.841
Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes
Romina H Aspera-Werz, Sabrina Ehnert, Monja Müller, Sheng Zhu, Tao Chen, Weidong Weng, Johann Jacoby, Andreas K Nussler
Romina H Aspera-Werz, Department of Traumatology, BG Trauma Clinic, Siegfried Weller Institute for Trauma Research, Eberhard Karls Universität Tübingen, Tübingen 72076, Germany
Sabrina Ehnert, Monja Müller, Sheng Zhu, Tao Chen, Weidong Weng, Andreas K Nussler, Department of Traumatology, BG Trauma Clinic, Siegfried Weller Institute for Trauma Research, Eberhard Karls Universität Tübingen, Tübingen 71076, Germany
Johann Jacoby, Institute for Clinical Epidemiology and Applied Biometry, Eberhard Karls Universität Tübingen, Tübingen 71076, Germany
Author contributions: Aspera-Werz RH, Ehnert S, and Nussler AK designed and coordinated the study; Müller M, and Aspera-Werz RH performed the experiments, acquired and analyzed data; Aspera-Werz RH, Ehnert S, Müller M, Jacoby J, and Nussler AK interpreted the data; Aspera-Werz RH wrote the manuscript; Ehnert S, Müller M, Zhu S, Chen T, Weng W, Jacoby J, and Nussler AK made critical revisions to the manuscript; all authors approved the final version of the article.
Institutional review board statement: This study was conducted under ethical approval by committee at the Medical Faculty of Eberhard-Karls-University Tübingen, No. 538/2014BO2.
Informed consent statement: All patients gave their written informed consent to participate in the study.
Conflict-of-interest statement: The authors have not conflict of interest.
Data sharing statement: The data used to support the findings of this study are available from the corresponding author upon reasonable request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Andreas K Nussler, Dr. rer.nat., Professor, Department of Traumatology, BG Trauma Clinic, Siegfried Weller Institute for Trauma Research, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, Tübingen 71076, Germany.
Received: February 25, 2020
Peer-review started: February 25, 2020
First decision: May 26, 2020
Revised: July 17, 2020
Accepted: August 1, 2020
Article in press: August 1, 2020
Published online: August 26, 2020

Cigarette smoking (CS) is the most common method of consuming tobacco. Deleterious effects on bone integrity, increased incidence of fractures, and delayed fracture healing are all associated with CS. Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and, via the bloodstream, reach the skeletal system. New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems, such as e-cigarettes and tobacco heating systems (THS). THS are designed to heat tobacco instead of burning it, thereby reducing the levels of harmful toxic compounds released.


To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS.


Human immortalized mesenchymal stem cells (n = 3) and primary human pre-osteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen (n = 5) were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4 “IQOS” or conventional “Marlboro” cigarettes for up to 21 d. Cell viability was analyzed using resazurin conversion assay (mitochondrial activity) and calcein-AM staining (esterase activity). Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase (AP) activity, while matrix formation was analyzed through alizarin red staining. Primary cilia structure was examined by acetylated α-tubulin immunofluorescent staining. Free radical production was evaluated with 2’,7’-dichlorofluorescein-diacetate assay.


Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity (P < 0.001). No significant differences in cytotoxicity between the diverse flavors of THS were observed. Harmful effects from THS on bone cell function were observed only at very high, non-physiological concentrations. In contrast, extracts from conventional cigarettes significantly reduced the AP activity (by two-fold) and matrix mineralization (four-fold) at low concentrations. Additionally, morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells, nor in the number of ciliated cells following THS treatment. Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells.


THS was significantly less harmful to osteoprogenitor cells during osteogenesis than conventional CS. Additional studies are required to confirm whether THS is a better alternative for smokers to improve delays in bone healing following fracture.

Keywords: Primary human osteoblast, Cigarette smoke, Tobacco heating system, Mesenchymal stem cells, Electronic nicotine delivery systems, Bone

Core tip: Aqueous extracts (AE) generated with tobacco heating systems (THS) showed no differences in suspended particles compared to the cell culture medium. This finding supports previous studies demonstrating reduced levels of harmful constituents reported in THS AE in comparison to conventional cigarettes AE. The time to consume one unit (stick/cigarette) was longer for THS than for a conventional cigarette. The pH from both AE fractions was similar to the cell culture medium. Following a single exposure, THS AE was significantly less toxic to bone-forming cells and osteoprogenitor cells than conventional cigarettes AE. The cytotoxicity observed following THS exposure was associated with very high, non-physiological concentrations. No significant differences in cytotoxicity were observed between different flavors of THS AE. Moreover, THS AE displayed less impact on osteogenic differentiation of osteoprogenitor cells and the function of bone-forming cells when compared to conventional cigarettes AE. Finally, compared to conventional cigarettes AE, THS AE induced lower levels of oxidative stress due to the reduced level of harmful constituents, resulting in less damage to primary cilia structure and reduced impact on osteogenic differentiation.